Systems and methods for angiogenic treatment in wound healing

ABSTRACT

The invention relates to systems and methods for the diagnosis, amelioration, and treatment of ischemic tissues in patients caused by and/or resulting from diminished microvascular blood flow. Patients suffering from ischemic tissue conditions can be categorized into specific subsets that are deemed to have a potential to respond to therapy. In particular, the invention includes various therapies involving stimulation of angiogenesis, vasculogenesis, arteriogenesis and/or neovascularization so as to increase perfusion of various tissues.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation patent application of U.S. patentapplication Ser. No. 14/573,153 filed Dec. 17, 2014, which is acontinuation patent application of U.S. patent application Ser. No.12/076,846 filed Mar. 24, 2008, now U.S. Pat. No. 8,983,570 issued onMar. 17, 2015, which is a non-provisional application of U.S.Provisional Application Ser. No. 61/022,266 filed Jan. 18, 2008 and U.S.Provisional Application Ser. No. 60/920,254 filed Mar. 27, 2007, theentire contents of which are incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

Embodiments described herein pertain to the field of diagnosing andtreating the spine, particularly lower back pain. Embodiments relate tomethods for diagnosing and/or treating disorders causative, or beingprecursors to back pain. In particular embodiments relate to methods ofincreasing angiogenesis in specifically diagnosed conditions associatedwith back pain.

STATEMENT OF FEDERALLY FUNDED RESEARCH

None.

INCORPORATION-BY-REFERENCE OF MATERIALS FILED ON COMPACT DISC

None.

BACKGROUND OF THE INVENTION

Musculoskeletal disorders of the spine are an extremely commonoccurrence associated with debilitating back pain, leading to enormouspsychosocial and economic ramifications. Lower-back pain is the leadingsource of disability in people under 45 years of age, and it results insignificant economic losses (Frymoyer J W, et al. The Adult Spine:Principles and Practice, 143-150, 1997). Eighty percent of people in theUnited States will experience back pain at some point in their lifetime(Lively, M. W., South Med J, 95:642-646, 2002), and it is the secondmost common reason for symptomatic physician visits (Hart, L. G. et al.Spine 20:11-19, 1995). Causes of back pain range from injury, whichpresents as a minor problem, accelerating to a chronic disorder, as wellas degenerative spine diseases that lead to degenerativespondylolisthesis and spinal stenosis. The vast majority of chronic backpain is associated with degeneration of the intervertebral disc, whichcan manifest in many different clinical conditions including spinalstenosis and instability, radiculopathy, myelopathy, and discherniation. In order to attain proper understanding of spinal pain,particularly lower back pain, we will review some of the anatomy of thespine, particularly the lumbar area.

The human spine is composed of bony structures called vertebrae,separated by intervertebral discs. One of the main functions of thevertebrae is to provide structural support and protection for the spinalcord. Each vertebra includes a spinous process, a bony prominence behindthe spinal cord, which shields the cord's nervous tissue on the backside, two bony protrusions on the sides called transverse processes, anda “body” in front of the spinal cord which provides a structural supportfor weight bearing. The average adult has 24 vertebrae, although atbirth 33 are present. Reduction in the number of vertebrae is caused byfusion during normal development. The vertebrae are divided byanatomical locations with 7 in the neck, also called the cervicalvertebrae, 12 in the middle back, called the thoracic vertebrae, 5 inthe lower back, called the lumbar vertebrae, and the sacrum, which isactually formed from five fused vertebrae. The tailbone, called thecoccyx is made of three fused vertebrae. Of these, the lumbar vertebraeare the largest, in part since they are responsible for carrying themajority of body weight. Consequently, the lumbar area is associatedwith the highest level of degeneration and is believed causative for awide variety of pain-inducing syndromes.

Separating the vertebrae are soft intervertebral discs that, togetherwith the two facet joints, allow for movement of the vertebrae andtherefore provide the ability of the spine to move in variousdirections. The complex of two facet joints posteriorly and the discanteriorly is referred to as a spinal segment. The intervertebral discincludes the annulus fibrosus, the nucleus pulposus, and the cartilageendplate. The nucleus pulposus includes anionic proteoglycans, such asaggrecan, that have high affinity for water, and provide a cushioningand shock-absorbing function. The annulus fibrosus encapsulates thenucleus pulposus, and is composed of concentrically organized layers ofcollagen fibrils (lamellae). The composition of the nucleus pulposus isdistinctly different than the annulus fibrosus since the formerprimarily includes a jelly-like substance and high collagen type I,whereas the latter is made of a solid, fibrotic-like texture, primarilycontaining collagen type II. In an adult, the cartilage endplate iscomposed primarily of hyaline cartilage and serves to separate thenucleus pulposus and annulus fibrosus from the adjacent vertebral bone.Discogenic pain often arises from areas of the annulus fibrosus. As amatter of fact, pain-associated molecules such as Substance P and TNF(Tumor Necrosis Factor) have been identified in large concentrations inbiopsy samples of patients suffering discogenic pain, but not incontrols.

Each disc provides motion and binds the segments together through its95% non-cellular and 5% cellular components. Nutrition to the disc cells(or chondrocytes) is provided by arteries that branch off of the majorartery in the body (called the aorta) and wrap around each vertebralbody, penetrating the bone along its circumference. The arteries coursethrough the vertebral body (bone) and then turn towards the disc ateither end. The oxygen, glucose and other nutrients are “dropped off”where the disc attaches to the bone and the capillaries and venules forma vascular loop. The cartilage that is in contact with these vascularloops is called the “endplate.” The nutrients “diffuse” (or move throughthe endplate and disc tissue without being transported in blood vessels)into the middle of the disc (or nucleus). In addition to this pathway,arterioles deliver nutrients to the outer edge of the disc (annulus)directly (this pathway provides minimal nutrients to the nucleus innormal discs but might be exploited in angiogenic treatment). Once thenutrients reach the cell, they are taken up and utilized for themanufacture of the materials that make up the disc (extracellularmatrix: collagen and proteoglycans). Recent studies have demonstratedthat cartilage cells require oxygen to produce enough energy for theproper manufacture and maintenance of the extracellular matrix. If thecells do not receive enough oxygen, the manufacturing process diminishesand the disc becomes acidic (pH drops) (Homer, H et al. Spine26:2543-2549, 2001). As the nutrient supply is cut off, the cells in thedisc begin to die and the disc tissue begins to breakdown. This loss ofnutrients is thought to be the initial cause of degenerative discdisease. As the disc continues to degenerate and the cell populationdecreases, the oxygen concentration may return to normal due to lessdemand. At this stage, regeneration still may be a possibility. However,excessive mechanical loading on a weakened structure precipitatesfurther degeneration which may lead to structural defects such asendplate fracture, radial fissures and herniation. As cells continue toproduce energy through anaerobic processes, low pH creates further celldeath.

This nutrient supply can be blocked at various stages. The feedingarteries themselves can narrow due to atherosclerosis with resultantischemia of the vertebral body. With less blood flowing through thevertebrae, less oxygen and nutrients are available to diffuse into thedisc creating hypoxia, lower pH and cell death (Kauppila, L et al. Spine22:1642-1649, 1997; Kurunlahit, M et al. Radiology 221:779-786, 2001).In addition to narrowing of the major lumbar vessels, many studies havedemonstrated decreasing blood flow within the vertebral body as thereason for the loss of nutrients and degenerative disc disease.Degenerative disc disease due to nicotine and aging demonstrate a lossof nutritive blood vessels in the area supplying nutrients (Iwahahi, Met al. Spine 27:1396-1401, 2002; Boos, N et al. Spine: 27:2631-2644,2002). Eventually, the endplate itself can become a hindrance to thediffusion of nutrients creating another obstacle to proper discchondrocyte nutrition (Rajasekaran, S et al. Spine 29:2654-2667, 2004).

Although disc degeneration continues to have a tremendous andever-increasing impact worldwide, current treatment options do notaddress the underlying cause. Current treatments include bed rest,nonsteroidal anti-inflammatory medication in the early phases ofpathology, and procedures such as discectomy, arthroplasty, (jointreplacement), injection of artificial nucleus pulposus and fusion in thelater phases when the prior approaches did not ameliorate pain. Suchapproaches are unpredictable, and deal almost exclusively with end-stageclinical manifestations, and therefore do nothing to alter the diseaseprocess itself. Additionally, procedures such as vertebral fusion resultin the increased incidence of disc degeneration in the adjacent discsdue to alterations in the biomechanical distribution of work-load.

Recent advances in both biotechnology and our understanding of thebiochemical makeup and environment of the intervertebral disc have ledto increased interest in the process of degeneration and the possibilityof developing novel treatments aimed directly at disc preservation.Certain genes found to have significant impact on matrix synthesis andcatabolism within the disc have provided targets for scientists seekingto alter the balance between the two. To this end, much attention overthe past several years has centered on gene therapy, and these effortshave yielded some promising preclinical results with regard to its usein treating disc degeneration (Levicoff, E. A. et al. Spine J5:2878-2968, 2005). Unfortunately, none of these approaches are nearclinical implementation at the time of this writing. Additionally, it isimportant to note that even in the circumstance that disc regenerationalone can be achieved through gene therapy or other interventionalmeans, the underlying process that originally caused the degenerationmust be addressed in order to prevent recurrence.

Currently, no biological treatment is widely available for discdegeneration. However, many different molecules of potential therapeuticbenefit are being investigated. The focus of molecular therapy has beento prevent or reverse one or more aspects of these changes in the discextracellular matrix. At least four different classes of molecules maybe effective in disc repair. These include anticatabolics, mitogens,chondrogenic morphogens and intracellular regulators (Yoon, S. T. SpineJ 5:2808-2868, 2005; Masuda, K. et al. Spine 29:2757-2769, 2004; Shimer,A. L. et al. Spine 29:2770-2778, 2004). Hallmarks of disc degenerationinclude loss of proteoglycans, water, and Type II collagen in the discmatrix. Other changes in the matrix are less well defined, includingloss of the higher molecular weight proteoglycans, collagencross-linking and organization of the proteoglycan, etc. An importantprocess in disc degeneration seems to be the change of thedifferentiated chondrocyte phenotype in the nucleus pulposus into a morefibrotic phenotype. Together these changes in the disc matrix lead toalterations of the disc and vertebral anatomy that ultimately areassociated with a pathologic condition (Setton, L. A. et al. Spine29:2710-2723, 2004; Roughley, P. J. Spine 29:2691-2699, 2004).

Due to the fact that matrix loss is a balance between matrix synthesisand degradation, it is possible to increase disc matrix by increasingsynthesis or by decreasing degradation. One approach is to preventmatrix loss by inhibiting the degradative enzymes. Degenerated discshave elevated concentrations of matrix metalloproteinases (MMPs). Withinthe matrix, MMP activity is normally inhibited by tissue inhibitors ofMMPs (TIMPs) (Roberts, S. et al. Spine 25:3005-3013, 2000; Nagase, H. etal. J Biol Chem 274:21491-21494, 1999; Wallach, C. J. et al. Spine28:2331-2337, 2003). Wallach, et al. (Spine 28:2331-2337, 2003) testedwhether one of these anticatabolic molecules, TIMP-1, could increase theaccumulation of matrix proteoglycans with in vitro experiments. It wasobserved that TIMP-1 expression in disc cells increased accumulation andalso increased the “measured synthesis rate” of proteoglycans (Wallach,et al. Spine 28:2331-2337, 2003). Chondrogenic morphogens are cytokinesthat not only possess mitogenic capability but are characterized bytheir ability to increase the chondrocyte-specific phenotype of thetarget cell. Most of the research in chondrogenic morphogens has beenperformed with transforming growth factor-.beta. (TGF-.beta.), bonemorphogenetic proteins (BMPs) or growth and differentiation factors(GDFs). Chondrogenic morphogens are particularly attractive because theymay reverse the fibrotic phenotype of disc cells to the morechondrocytic phenotype of disc nucleus cells in younger and more“normal” discs. By definition, these molecules are secreted moleculesand hence can potentially act in autocrine, paracrine and endocrinefashion.

BMP-2 is another prototypic chondrogenic morphogen (Thompson, J. P. etal. Spine 16:253-260, 1991). Yoon, et al. (Spine 29:2603-2611, 2004)reported that recombinant human BMP-2 increased production of rat disccell proteoglycans and significantly increased the chondrocyticphenotype of the disc cells as shown by increased aggrecan and Type IIcollagen gene expression, whereas there was no change in Type I collagengene expression. Kim, et al. (J Neurosurg 99:291-297, 2003) reportedthat BMP-2 can partially reverse the inhibitory effect of nicotine onthe synthesis of disc cell proteoglycan. BMP-7, also known as OP-1(Osteogenic Protein-1), is another disc cell morphogen that hasdemonstrated potent in vitro activity in terms of enhancing matrixformation in disc cells (Masuda, K. et al. J Orthop Res 21:922-930,2003; Zhang, Y. et al. Am J Phys Med Rehabil 85:515-521, 2004; Takegami,K. et al. Spine 27:1318-1325, 2002). Growth differentiation factor 5(GDF-5) is also known as CDMP-1 (Cartilage-derived morphogeneticprotein 1) and has also been considered for regeneration of disc cells.However, only in vitro experimentation has been performed to date(Chang, S. C. et al. J Biol Chem 269:28227-28234, 1994).

Intracellular regulators are a distinct class of molecules because theyare not secreted and do not work through transmembrane receptors. Thesemolecules are neither cytokines nor growth factors in the classicalsense, and yet they can have effects that are quite similar to thesecreted molecules discussed previously. This class of moleculestypically controls one or more aspects of cellular differentiation. Forinstance, Sma-Mad (SMAD) proteins are intracellular molecules thatmediate BMP-receptor signaling (Nohe, A. et al. Cell Signal 16:291-299,2004; Hatakeyama, Y. et al. J Bone Joint Surg Am 85-A Suppl 3:13-18,2003). Although there are no specific published papers on the effect ofSMAD proteins on disc cells, proteins such as Smad-1 and Smad-5 arepredicted to induce similar effects on disc cells as BMP-2, such asincreasing proteoglycan and Type II collagen synthesis. Sox9(transcription factor) is a chondrocyte marker that is a positiveregulator of Type II collagen mRNA transcription (Yoon, S. T. Spine J5:280 S-2865, 2005; Li, Y. et al. Tissue Eng 10:575-584, 2004; Aigner,T. et al. Matrix Biol 22:363-372, 2003). Paul, et al. (Spine 28:755-763,2003) showed that Sox9 delivered by adenovirus can increase Sox9expression and disc cell production of Type II collagen in in vitroexperiments.

The success of a disc tissue engineering strategy is dependent onmolecular cues to direct the differentiation of cells and affect theirbiosynthetic function. Many growth factors, including members of thetransforming growth factor beta superfamily, affect the differentiationprocess of disc cells. This group of related proteins directs theinduction of mesenchymal precursors to form mature skeletal tissues(Sampath, T. K. et al. Proc Natl Acad Sci USA 81:3419-3423, 1984). Theactivity of these molecules is complex and affects intercellularsignaling pathways (Israel, D. I. et al. Growth Factors 13:291-300,1996; Heldin, C. H. et al. Nature 390:465-471, 1997). In addition,concentration and timing of presentation of the growth factor influencesits activity. Depending on the tissue, the effects of a given morphogenmay be different. For instance, the osteogenic molecule bonemorphogenetic protein-7-osteogenic protein-1 (BMP-7/OP-1) has been shownto have a dramatic effect on disc cells, increasing their metabolicoutput of matrix proteins and rescuing them from the detrimental effectsof Interleukin 1 (IL-I) (Takegami, K. et al. Spine 27:1318-1325, 2002).This data suggests that growth factors could play a useful role in acell-based tissue engineering strategy.

Other yet to be identified factors direct cell-to-cell communication andappear to play an important role in the viability and metabolic activityof disc cells. Yamamoto, et al. (Spine 29:1508-1514, 2004) showed thatcell proliferation and proteoglycan synthesis was significantly enhancedin disc cells cultured in a system that allowed direct cell-cell contactwith bone marrow-derived stromal cells. In another study, Hunter, et al.(Spine 29:1099-1104, 2004) reported that enzymatic disruption of gapjunctions produced a negative effect on cell viability, suggesting thatcommunication among adjacent cells plays a vital role in cellularviability and function, and therefore interventions supporting theirenhancement may be beneficial.

The invention relates to methods for diagnosing, treating orameliorating painful conditions of the spine, particularly lower backpain. Embodiments are directed to classification of back pain that isbased on specific parameters associated with ischemia, which is adecrease blood flow inflow due to arterial blockage, hypoperfusion,which is diminished microvascular blood flow, and the resulting hypoxia,which is decreased oxygen within the tissue, with resultant damage totissue. Further embodiments relate to treatments for alleviating thestate of ischemia, hypopersion and hypoxia in patients that may lead totherapeutic improvement.

SUMMARY OF THE INVENTION

In an embodiment, diagnosis of ischemic or hypoxic disc disease, oftendescribed as “lumbar ischemia,” as a disorder is made by the two-parttest of first excluding patients with a set of exclusion criteria, andfurther selecting patients having documented ischemia, hypoperfusion,and/or hypoxia, of the affected areas. Specific exclusion criteriainclude, for example, the presence of herniated disc, spinal infection,spinal tumor, spinal arthritis, and spinal canal stenosis.

Embodiments of the invention are directed to methods of diagnosing acondition responsible for degenerative disc disease or vertebralosteoporosis, which may include one or more of the following steps:

-   -   a) assessing a patient by one or more of the following steps:    -   (i) classifying patency of said one or more lumbar segmental        vessels;    -   (ii) determining blood perfusion in the anatomical areas        supplied by said segmental vessels;    -   (iii) determining extent of disc degeneration or vertebral        osteoporosis;    -   b) correlating data collected from a(i) with data collected from        (a(ii) and with data collected from a(iii);    -   c) producing an overall index of correlation; and    -   d) comparing said index of correlation with an index of        correlation generated from a healthy population.

In one embodiment, hypoxic and/or ischemic disc disease is treated byincreasing perfusion in the affected area such as by injection of acomposition that includes an angiogenic factor. In preferredembodiments, injection is around the vertebrae or directly into thevertebral body. In other embodiments, a localized delivery systemcapable of forming a gel-like structure may be used to deliver theangiogenic factor. Preferably, the delivery system includes componentsof extracellular matrix that provide conditions suitable forangiogenesis. In other embodiments, the extracellular matrix componentsmay be hyaluronic acid fragments. In other embodiments, theextracellular matrix components may be derivatives of collagen, orperlecan. Preferably, the gel-like structure includes a polymer capableof slow release such as a poloxamer block copolymer (Pluronic®, BASF),basement membrane preparation (Matrigel®, BD Biosciences) orcollagen-based matrix such as described in U.S. Pat. No. 6,346,515,which is incorporated herein by reference in its entirety.

In another embodiment, hypoxic and/or ischemic disc disease is treatedby administration of a medical device that generates a continuousrelease of a composition that includes an angiogenic factor into tissueand/or circulation so as to promote neoangiogenesis, and specifically,collateralization in the area(s) proximal to hypoperfusion. In someembodiments, the composition further includes stem cells. The medicaldevice may include a slow release pump such as an implantable indwellingor osmotic pump or a localized delivery system such as a polymer capableof slow release as described above.

In another embodiment, the composition delivered by the medical deviceincludes both a therapeutically sufficient concentration of a growthfactor that stimulates angiogenesis and a chemotactic agent. Some growthfactors, such as fibroblast growth factor 1 (FGF-1), are themselveschemotactic. The chemotactic agent recruits cells capable of causing orpromoting angiogenesis. In some embodiments, a chemotactic agent such asstromal cell-derived factor 1 (SDF-1) is included in the compositionwith the growth factor.

Assessment of perfusion followed by therapy that increases the rate ofperfusion, followed by a subsequent assessment of perfusion so as toidentify the ideal conditions for stimulation of perfusion on anindividualized basis.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are included to provide a furtherunderstanding of the invention and are incorporated in and constitute apart of this specification, illustrate embodiments of the invention andtogether with the description serve to explain the principles of theinvention. In the drawings:

FIG. 1 represents coronal MRA (magnetic resonance angiography) and T2weighted sagittal MM (magnetic resonance imaging) for a healthy controlsubject compared to 3 subjects (Sub1, Sub2, Sub3) with symptoms ofchronic lower back pain and degenerative disc disease (DDD). Also shownis a Max Intensity Projection (MIP) and Axial reconstruction for Sub3.The control is a 20-year old subject with normal discs and segmentalvessels from L1 to L5. The arrows indicate areas of stenosis orocclusion. Sub1 shows areas of stenosis indicated by the arrows. Sub2shows areas of occlusion indicated by the arrow. Sub3 shows areas ofboth occlusion (left arrow) and stenosis (right arrow);

FIGS. 2A through 2D show data for subject 3 (sub3) of FIG. 1. FIG. 2Ashows an abnormal MRA scan with areas of both occlusion (left—whitecircle) and stenosis (right—white arrow). FIG. 2B shows Grade 1 DDD.FIG. 2C shows pixilation of perfusion data. FIG. 2D shows a bar forcomparison, where the shading at the top of the bar is maximumK.sup.trans. The side arrow shown in FIG. 2D indicates that theperfusion data of FIG. 2C shows relatively low flow of blood throughvertebrae;

FIGS. 3A through 3D show data for the control of FIG. 1. FIG. 3A shows anormal MRA scan. FIG. 3B shows normal discs. FIG. 3C shows pixilation ofperfusion data. FIG. 3D shows a bar for comparison, where the shading atthe top of the bar is maximum Ktrans. The side arrow shown in FIG. 3Dindicates that the perfusion data of FIG. 3C shows relatively high flowof blood through vertebrae;

FIG. 4A shows a Max Intensity projection (MIP) and FIG. 4B shows anAxial reconstruction for sub3. The circle in FIG. 4A and the right-sidecircle in FIG. 4B point to an occluded vessel. The left hand arrow inFIG. 4B points to a stenosis;

FIG. 5A shows MR (magnetic resonance) spectroscopy of the L4-5 disc andFIG. 5B shows MR spectroscopy of the L5-1 disc. FIG. 6A shows thewater-unsuppressed spectra of FIG. 5A and FIG. 5B. FIG. 6B shows thesame spectrum as FIG. 6A but with water suppressed. “Lip”=lipid.“Lac”=lactate.

DETAILED DESCRIPTION OF THE INVENTION

Although some embodiments are described below, these are merelyrepresentative and one of skill in the art will be able to extrapolatenumerous other applications and deviates that are still within the scopeof the invention disclosed.

Numerous studies suggested the notion that the vast majority of patientswith long-term back pain, intractable by conventional approaches, haveoccluded lumbar/middle sacral arteries and that occlusion of thesearteries is associated with disc degeneration (Kauppila, L. I. et al.Spine 29:2147-2152, 2004). However, none of these studies proposedtreatment methodologies, or placed it in the context of disease-specificdiagnosis. These studies have included observations that patients withhigh LDL (low density lipoprotein) cholesterol complained of more severeback symptoms than those with normal value (Kauppila, L. I. et al. Spine29:2147-2152, 2004). These findings support previous studies thatocclusion of lumbar/middle sacral arteries is associated with lower backpain and disc degeneration (Kauppila, L. I. Lancet 346:888-889, 1995;Boggild, H. Scand J Work Environ Health 32:20-21, 2006; Kauppila, L. I.et al. J Spinal Disord 6:124-129, 1993; Kauppila, L. I. et al. Spine22:1642-1647, 1997; Kurunlahti, M. et al. Spine 24:2080-2084, 1999) andthat occlusion of these arteries may be due to atherosclerosis (Cluroe,A. D. et al. Pathology 24:140-145, 1992; Kauppila, L. I. et al. Spine19:923-929, 1994). Epidemiologic and post-mortem studies indicate thatatheromatous lesions in the abdominal aorta may be related to discdegeneration and long-term back symptoms (Kauppila, L. I. Lancet346:888-889, 1995; Boggild, H. Scand J Work Environ Health 32:20-21,2006; Kauppila, L. I. et al. J Spinal Disord 6:124-129, 1993; Kauppila,L. I. et al. Spine 22:1642-1647, 1997; Kurunlahti, M. et al. Spine24:2080-2084, 1999)). However, these studies have not provided a meansfor classification of patients, or for therapeutic interventions.Additionally, since disc degeneration is not necessarily a painfulprocess, the relevance of occluded spinal arteries remains enigmatic.

Embodiments described herein provide hypoxic and/or ischemic discdisease as a defined disease subset, in which patients may bespecifically classified that are amenable to treatment with meanscapable of stimulating perfusion. Specifically, in one embodiment,hypoxic and/or ischemic disc disease is diagnosed as stenosis or thecomplete occlusion of one or more blood vessels associated with thelumbar area. The importance of perfusion is seen in the followingdiscussion regarding lumbar vasculature. It is known that the bloodsupply of the lumbar spine is derived from the aorta through the lumbarand middle sacral arteries. The upper four segments of the lumbar spinereceive their blood supply from the four pairs of the lumbar arteries,which arise in the posterior wall of the abdominal aorta. The fifthlumbar segment is supplied partly by the middle sacral artery (arisingin the bifurcation) and partly by branches of the iliolumbar arteries(arising from the internal iliac arteries) (Crock, H. V. The Practice ofSpinal Surgery, Springer-Verlag, 1983); Kauppila, L. I. Acta Radiol35:541-544, 1994). Nutrition of the avascular intervertebral disc occursby diffusion through the vertebral endplates from the blood vessels inthe vertebral bodies above and below the disc (Urban, J. P. et al. Spine29:2700-2709, 2004; Walker, M. H. et al. Spine J 4:158S-166S, 2004).Cholesterol plaques in the wall of the aorta obliterate orifices oflumbar and middle sacral arteries and decrease blood supply of thelumbar spine and its surrounding structures. As a result, structureswith precarious nutrient supply, such as the intervertebral discs,gradually degenerate (Cluroe, A. D. et al. Pathology 24:140-145, 1992;Mitchell, J. R. et al. Atherosclerosis 27:437-446, 1977; Ross, R.Cecil's Textbook of Medicine 318-23, 13, 1988). Reduced blood flowcauses hypoxia and tissue dysfunction. It also hampers removal of wasteproducts, such as lactic acid. These changes are found by the currentinvention to mean that in some patients irritation of nociceptive nerveendings occurs, causing pain, as well as lead to deterioration andatrophy of the structures involved (Naves, L. A. et al. Braz J Med BiolRes 38:1561-1569, 2005; Iwabuchi, M. et al. Spine 26:1651-1655, 2001;Ohshima, H. et al. Spine 17:1079-1082, 1992; Bibby, S. R. et al. EurSpine J 13:695-701, 2004). Accordingly, the invention provides both amethod of quantifying and relating hypoperfusion with pathological andsymptomatic features and also methods of selecting patients that wouldbenefit from interventions aimed at stimulating perfusion in the area ofthe spine, or areas associated with not only lack of blood supply butalso removal of metabolic wastes.

In one embodiment of the invention, a patient is diagnosed with hypoxicand/or ischemic disc disease and is treated by increasing localizedperfusion through the use of angiogenesis induction. The process of newblood vessel formation (angiogenesis) can occur naturally, or be inducedthrough three various means, namely: vasculogenesis, arteriogenesis, andangiogenesis. For the purpose of this invention, all three will bereferred to as “angiogenesis.” Angiogenesis is associated with de novocapillary formation from post-capillary venules, is hypoxia-driven, andis associated with a 2-3 fold increase in blood flow. Arteriogenesis isthe remodeling of pre-existing vascular channels (collaterals) or denovo artery formation. It is stimulated by local changes in perfusion(shear stress), as well as cellular influx, and associated with a 20-30fold increase in blood flow. Vasculogenesis is responsible for embryonicvascular development and includes de novo formation of vascular channelsinitiated by circulating vascular precursor cells. Furthermore, it isconsidered to be ischemia and injury initiated (Simons, M. Circulation111:1556-1566, 2005). The term “angiogenesis” is used in thisapplication to encompass all three technical terms due to the functionaluncertainty at present regarding the continuum of biological andphysiological differences among these sub-divisions.

Angiogenesis is known to occur physiologically duringimplantation/embryogenesis (Sharkey, A. M. et al. Contraception71:263-271, 2005), wound healing (Dvorak, H. F. J Thromb Haemost3:1835-1842, 2005), and expansion of adipose mass (Voros, G. et al.Endocrinology 146:4545-4554, 2005). Pathologically, uncontrolledangiogenesis is associated with a variety of diseases such as maculardegeneration (Kroll, P. et al. Br J Ophthalmol 90:128-130, 2006), cancer(Folkman, J. Semin Oncol 29:15-18, 2002), arthritis (Maruotti, N. et al.Histol Histopathol 21:557-566, 2006), and atherosclerosis (Conway, E. M.Pathophysiol Haemost Thromb 33:241-248, 2003). One common aspect ofadult angiogenesis is the issue of tissue hypoxia. In all situations oftissue expansion, cells are dependent on the microvasculature fornutrients and oxygen supply, as well as removal of metabolic wasteproducts. Accordingly during tissue growth, cells begin to “sense” alack of oxygen. This triggers a cascade of events that culminates inangiogenesis. During pathological conditions, such as the conditionsassociated with hypoxic and/or ischemic disc disease, the lack of oxygenis induced through hypoperfusion. The hypoperfusion may occur due to,for example, atherosclerosis. In some pathological conditions, thenormal angiogenic response to hypoxia is absent or substantiallydiminished.

Although numerous methods of physiological stimulation of angiogenesisunder hypoxia are known and thereby useful for the practice of thecurrent invention (Mizukami, Y. et al. J Biol Chem 2006), one of themost well characterized pathways involves activation of the HypoxiaInducible Factor-1 (HIF-1), transcription factor (Liu, L. et al. CancerBiol Ther 3:492-497, 2004). This protein is only functionally active asa heterodimer of HIF-1.alpha. and HIF-1.beta., which are both basichelix-loop-helix proteins. While the latter is known to be relativelystable, the former has a half-life of less than 5 minutes underphysiological conditions due to rapid proteasomal degradation by theoxygen sensitive von Hippel-Lindau (VHL) E3-ubiquitin ligase system(Ivan, M. et al. Science 292:464-468, 2001). When cells experiencehypoxia, HIF-1.alpha. half-life is increased since the degradation byVHL E3-ubiquitin ligase is dependent on proline hydroxylation, whichrequires molecular oxygen. Therefore, this protein modification plays akey role in mammalian oxygen sensing. Activation of this transcriptionfactor leads to gene expression of numerous angiogenesis related genessuch as VEGFs (Gray, M. J. et al. Oncogene 24:3110-3120, 2005), FGF-2response genes (Li, J. et al. J Cell Sci 115:1951-1959, 2002), notchsignaling (Pear, W. S. et al. Cancer Cell 18:435-437, 2005), and upregulation of stromal derived factor (SDF-1), which chemoattractsendothelial precursors during angiogenesis (Ceradini, D. J. et al.Trends Cardiovasc Med 15:57-63, 2005). There are numerous variations bywhich angiogenesis can occur; however, the basic steps involveremodeling of the extracellular matrix through matrix metalloproteases(MMPs), chemoattraction of either precursor endothelial cells orexisting endothelial cells from an adjacent vessel, proliferation of theendothelial cells, tube formation and stabilization. Embodiments relateto transfection of genes encoding HIF-1 into areas of lumbarhypoperfusion in order to induce normalization of perfusion, or in somecases hyperperfusion in order to ameliorate or significantly treathypoxic and/or ischemic disc disease. Embodiments described hereinrelate to, among other things, utilization of molecules that eitherinduce the expression of HIF-1, or conversely delay the degradation ofHIF-1 or components thereof including but not limited to FGFs.

In one embodiment of the invention, the stimulation of perfusion in thearea proximal to the pain generator is performed by providing propernutrition so as to enhance healing and production of appropriateproteins in said disc. It is known that the synthesis of proteoglycansin the nucleus pulposus occurs naturally by the cellular component ofthe nucleus pulposus. Specific growth factors such as transforminggrowth factor-.beta. (TGF-.beta.) and epidermal growth factor (EGF) areinvolved in the stimulation of proteoglycan synthesis. Interestingly, inpatients with degenerative disc disease, the amount of these cytokinesis reduced in comparison to healthy nucleus pulposus cells (Konttinen,Y. T. et al. J Bone Joint Surg Br 81:1058-1063, 1999). This reductionmay be due to decreased nutrient supply and cellular viability withinsaid nucleus. Another reason for inhibition of proteoglycan synthesis islower pH caused by ischemia and/or hypoperfusion of the lumbar area(Razaq, S. et al. Eur Spine J 12:341-349, 2003). The low pH also appearsto be involved in another process associated with discogenic pain, saidprocess comprising up regulation of matrix metalloproteases expression.It is known that matrix metalloproteases are involved in cleavingproteoglycans, and that up regulation of matrix metalloprotease activityis associated with disc degeneration (Gruber, H. E. et al. BiotechHistochem 80:157-162, 2005). Activation of matrix metalloproteases isknown to be induced by inflammatory cytokines such as TNF and IL-1(Seguin, C. A. et al. Spine 30:1940-1948, 2005). Additionally, animalstudies have demonstrated that hyperphysiological loading of the discsegment induces up regulation of matrix metalloproteases (OmLor, G. W.et al. J Orthop Res 24:385-392, 2006), but have not assessed theinfluence of perfusion. Accordingly, in one embodiment of the invention,the increase of localized perfusion is used to augment proteoglycancontent in said nucleus pulposus, as well as to lead to suppression, insome instances, of MMP activation.

In one embodiment, patients with advanced lumbar back pain are screenedto determine whether the pain is associated with disc degeneration. Thisscreening is a common medical practice and includes techniques such asphysical examination, radiographic studies, MM and bone scan to diagnose“discogenic” pain, or pain associated with degeneration of the annulusfibrosus, nerve irritation by the nucleus pulposus, or other chronicpain. Patients with rheumatoid arthritis, spinal infections or tumors,acute nerve compression and arthritis are excluded from eligibility fortreatment using the methods and compositions described in the presentinvention. In a variety of cases, patients treated with the inventiondisclosed will be refractory to conventional treatments such asanti-inflammatory medication or analgesics. In a more specificembodiment, patients are diagnosed based on degeneration of a single orplurality of discs using magnetic resonance imaging. In preferredembodiments, disc degeneration can be estimated from regular lumbar T1and T2-weighted MR sagittal fast spin-echo (FSE) and T2-weighted FSEaxial images. Preferably, the intervertebral discs may be classifiedaccording to three grades: grade 0, discs with high signal intensity oronly slightly blurred intranuclear cleft, which represent normal discs;grade 1, discs with decreased signal intensity but normal height, whichrepresent mild degeneration; and grade 2, discs with markedly decreasedsignal intensity and height loss, which represent severe degeneration.In preferred embodiments, the signal intensities of intervertebral discsare compared with those of cerebrospinal fluid. In some embodiments,specific grades of disc degeneration are chosen for treatment. In aparticularly preferred embodiment, parameters used for MR imaging ofdiscs includes: TR 3200 ms, TE 119 ms milliseconds, and thickness of 4.0mm with gap 0.4 mm.

Numerous methods are known to identify the disc segment causative oflower back pain, as well as areas of hypoperfusion. These includediffusion-weighted imaging, magnetic resonance imaging, diffusion tensorimaging, magnetic resonance spectroscopy, functional magnetic resonanceimaging, dynamic computed tomography and magnetic resonance imaging, T2relaxometry MRI, CT-scan (computed tomography scan), and provocativediscography (Haughton, V. J Bone Joint Surg Am 88 Suppl 2:15-20, 2006).Any of these techniques may be used alone or in combination to diagnoselumbar ischemia as described.

In one embodiment, the area of hypoperfusion is identified usingtechnetium-99m Sestamibi in conjunction with single photon emissioncomputed tomography (SPECT) imaging. This radiolabelled lipophiliccation is injected intravenously at concentrations ranging from 200-1790MBq, more preferably 500-1000 MBq, and even more preferable atapproximately 750 MBq. Imaging is performed with a gamma camera andabsorption/perfusion is quantified using various software packages knownto one skilled in the art. In some embodiments, to attain appropriateimages of the lumbar area, the camera is rotated 360 degrees.

In other embodiments, various other methodologies for detectinghypoperfusion are employed, for example, PET-CT (positron emissiontomography-computed tomography), MR diffusion angiography, and, forexample, fluorescent peptide-based methodologies.

Once an area of hypoperfusion is identified, and a patient is diagnosedwith hypoxic and/or ischemic disc disease, induction ofneovascularization is performed so as to enhance localized perfusion tothe area of need. In one embodiment, genes are introduced from exogenoussources so as to promote angiogenesis. It is known that genes may beintroduced by a wide range of approaches including adenoviral,adeno-associated, retroviral, alpha-viral, lentiviral, Kunjin virus, orHSV vectors, liposomal, nano-particle mediated as well aselectroporation and Sleeping Beauty transposons. Genes with angiogenicstimulatory function that may be transfected include, but are notlimited to: VEGFs, FGF-1, FGF-2, FGF-4, and HGF. Additionally,transcription factors that are associated with up regulating expressionof angiogenic cascades may also be transfected into cells used fortreatment of lower back pain. The genes include: HIF-1.alpha., HIF-2,NET (norepinephrine transporter gene), and NF-kB (nuclear factor-kappaB). Antisense oligonucleotides, ribozymes or short interfering RNA(ribonucleic acid) may be transfected into cells for use for treatmentof lower back pain in order to block expression of antiangiogenicproteins such as IP-10 (Interferon-gamma-inducible 10 kD protein).

Selection of genes or techniques for introduction of said genes in vivomay be performed in vitro prior to administration so as to allow formethods of screening and selecting the combination that is mostangiogenically potent. Testing may be performed by various methodologiesknown to one skilled in the art. In terms of assessing angiogenicpotential, these methodologies include, but are not limited to:

A. Angiogenic activity may by assessed by the ability to stimulateendothelial cell proliferation in vitro using human umbilical veinendothelial cells (HUVECs) or other endothelial cell populations.Assessment of proliferation may be performed using tritiated thymidineincorporation or by visually counting the proliferating endothelialcells. A viability dye such as MTT or other commercially availableindicators may be used;

B. Angiogenic activity may also be assessed by the ability to supportcord formation in subcutaneously implanted matrices. The matrices, whichmay include Matrigel® or fibrin gel, are loaded with cells that do nothave intrinsic angiogenic potential, for example fibroblasts,transfecting said cells with said genes, and implanting said cellssubcutaneously in an animal. The animal may be an immunodeficient mousesuch as a SCID (severe combined immunodeficiency) or nude mouse in orderto negate immunological differences. Subsequent to implantation,formation of endothelial cords generated from endogenous host cells maybe assessed visually by microscopy. In order to distinguish cellsstimulating angiogenesis versus host cells responding to the cellsstimulating angiogenesis, a species-specific marker may be used;

C. Angiogenic activity may be assessed by the ability to accelerateangiogenesis occurring in the embryonic chicken chorioallantoic membraneassay. Cells transfected with angiogenic genes may be implanteddirectly, or via a matrix, into the chicken chorioallantoic membrane onembryonic day 9 and cultured for a period of approximately 2 days.Visualization of angiogenesis may be performed using in vivo microscopy;and

D. Angiogenic activity may be assessed by the ability to stimulateneovascularization in the hind limb ischemia animal model. In oneembodiment, patients diagnosed with hypoxic and/or ischemic disc diseaseare treated using gene therapy in a localized manner.

In one embodiment, patients diagnosed with hypoxic and/or ischemic discdisease are treated using gene therapy in a localized manner.Specifically, the gene for FGF-1 is administered in a composition ofnucleic acid sequences or one or more triplex DNA compounds, and anonionic block copolymer. The gene administered is under control of astrong promoter, for example, the CMV (cytomegalovirus) promoter. Thenonionic block copolymer may be CRL-8131 as described in U.S. Pat. No.6,933,286 (which is incorporated herein by reference in its entirety).Specifically, 300 milligrams of CRL-8131 may be added to 10 mL of 0.9%NaCl and the mixture is solubilized by storage at temperatures of2-4.degree. C. until a clear solution is formed. An appropriate amountof a FGF-1 expressing plasmid diluted in PBS (phosphate buffered saline)is added to the mixture and micelles associating the copolymer and thecompound are formed by raising the temperature above .degree. C. andallowing the suspension of micelles to equilibrate. The equilibratedsuspension is suitable for administration.

In other embodiments, it may be desirable to utilize anangiogenesis-stimulating protein for administration in a therapeuticallyeffective amount. The protein may be selected from proteins known tostimulate angiogenesis including, but not limited to: TPO (thyroidperoxidase), SCF (stem cell factor), IL-I (interleukin 1), IL-3, IL-6,IL-7, IL-8, IL-11, flt-3L (fms-like tyrosine kinase 3 ligand), G-CSF(granulocyte-colony stimulating factor), GM-CSF (granulocytemonocyte-colony stimulating factor), Epo (erythropoietin), FGF-1, FGF-2,FGF-4, FGF-5, FGF-20, IGF (insulin-like growth factor), EGF (epidermalgrowth factor), NGF (nerve growth factor), LIF (leukemia inhibitoryfactor), PDGF (platelet-derived growth factor), BMPs (bone morphogeneticprotein), activin-A, VEGF (vascular endothelial growth factor), VEGF-B,VEGF-C, VEGF-D, P1GF, angiopoietins, ephrins, and HGF (hepatocyte growthfactor). In some preferred embodiments, administration of theangiogenesis-stimulating protein is performed by injection directly intoa vertebral body. In some embodiments, the angiogenic-stimulatingprotein is co-administered with stem or progenitor cells. The stem cellsmay be embryonic stem cells or adult stem cells.

As used herein, the terms “treating,” “treatment,” “therapeutic,” or“therapy” do not necessarily mean total cure or abolition of the diseaseor condition. Any alleviation of any undesired signs or symptoms of adisease or condition, to any extent, can be considered treatment and/ortherapy. In addition, asymptomatic degenerative disc disease may be thefocus of treatment utilizing angiogenesis. Furthermore, treatment mayinclude acts that may worsen the patient's overall feeling of well-beingor appearance.

The term “therapeutically effective amount” is used to indicate anamount of an active compound, or pharmaceutical agent, that elicits thebiological or medicinal response indicated. This response may occur in atissue, system, animal or human and includes alleviation of the symptomsof the disease being treated.

The exact formulation, route of administration and dosage for thecomposition and pharmaceutical compositions disclosed herein can bechosen by the individual physician in view of the patient's condition.(See, e.g., Fingl et al. 1975, in “The Pharmacological Basis ofTherapeutics”, Chapter 1, which is hereby incorporated herein byreference in its entirety). The dosage may be a single one or a seriesof two or more given in the course of one or more days, as is needed bythe patient. Where no human dosage is established, a suitable humandosage can be inferred from ED.sub.50 or ID.sub.50 values, or otherappropriate values derived from in vitro or in vivo studies, asqualified by toxicity studies and efficacy studies in animals.

Although the exact dosage will be determined on a drug-by-drug basis, inmost cases, some generalizations regarding the dosage can be made.Alternatively, the compositions disclosed herein may be administered bycontinuous infusion, preferably at a dose of each active ingredient upto approximately 400 ug per day. Thus, the total daily dosage byparenteral administration will typically be in the range ofapproximately 0.1 to approximately 400 ug. In some embodiments, thecompounds will be administered for a period of continuous therapy, forexample for a week or more, or for months.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety, which are sufficient to maintain themodulating effects, or minimal effective concentration (MEC). The MECwill vary for each compound but can be estimated from in vitro data.Dosages necessary to achieve the MEC will depend on individualcharacteristics and route of administration. However, HPLC(high-performance liquid chromatography) assays or bioassays can be usedto determine plasma concentrations.

Dosage intervals can also be determined using MEC value. Compositionsshould be administered using a regimen which maintains plasma levelsabove the MEC for approximately 10% and approximately 90% of the time,preferably between approximately 30% and approximately 90%, and mostpreferably between approximately 50% and approximately 90%.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

In some embodiments a carrier solution is desired. The carrier solutionsmay be selected based on properties such as viscosity, ease ofadministration, ability to bind solution over a period of time, andgeneral affinity for the agent delivered. The solutions may be modifiedor additives incorporated for modification of biological properties.Starting solutions may include certain delivery polymers known to onewho is skilled in the art. These could be selected from, for example:polylactic acid (PLA), poly-L-lactic acid (PLLA), poly-D-lactic acid(PDLA), polyglycolide, polyglycolic acid (PGA), polylactide-co-glycolide(PLGA), polydioxanone, polygluconate, polylactic acid-polyethylene oxidecopolymers, polyethylene oxide, modified cellulose, collagen,polyhydroxybutyrate, polyhydroxpriopionic acid, polyphosphoester,poly(alpha-hydroxy acid), polycaprolactone, polycarbonates, polyamides,polyanhydrides, polyamine acids, polyorthoesters, polyacetals,polycyanoacrylates, degradable urethanes, aliphatic polyesterpolyacrylates, polymethacrylate, acryl substituted cellulose acetates,non-degradable polyurethanes, polystyrenes, polyvinyl fluoride,polyvinyl imidazole, chlorosulphonated polyolefin, and polyvinylalcohol.

Administration may be performed under fluoroscopy or other means inorder to allow for localization in proximity of the cause ofhypoperfusion. Acceptable carriers, excipients, or stabilizers are alsocontemplated within the current invention; said carriers, excipients andstabilizers being relatively nontoxic to recipients at the dosages andconcentrations employed, and may include buffers such as phosphate,citrate, and other organic acids; antioxidants including ascorbic acid,n-acetylcysteine, alpha tocopherol, and methionine; preservatives suchas hexamethonium chloride; octadecyldimethylbenzyl ammonium chloride;benzalkonium chloride; phenol, benzyl alcohol, or butyl; alkyl parabenssuch as methyl or propyl paraben; catechol; resorcinol; cyclohexinol;3-pentanol; and mecresol; low molecular weight polypeptides; proteins,such as gelatin, or non-specific immunoglobulins; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA(ethylenediaminetetraacetic acid); sugars such as sucrose, mannitol,trehalose, or sorbitol; salt-forming counter-ions such as sodium.

In an embodiment, a localized medical device is implanted using anattachment means onto an anatomical structure that resides proximal tothe blood vessel supplying the area of hypoperfusion. In anotherembodiment, the attachment is performed using an anchoring means; saidanchoring means attaching said medical device to the vertebral boneproximal to one of the lumbar or medial sacral arteries. The medicaldevice includes the functionality of time-course release of anangiogenic factor. The medical device may be composed of a solid casingwith internal gel-like fluid containing desired angiogenic factor. Thegel-like fluid may be a cryoprecipitate, an administration matrix, or acomposition of various polymers suitable for the sustained release ofsaid angiogenesis promoting factor.

In another embodiment, treatment of hypoxic and/or ischemic disc diseaseincludes the use of a biodegradable implant. The biodegradable implantcontains a biodegradable delivery means, or carrier, as well asangiogenic factors; said angiogenic factors are capable of stimulatingsufficient neovascularization to overcome local hypoxia. One preferredangiogenic factor is fibroblast growth factor 1 (FGF-1). However, otherrecombinant naturally derived, in vitro derived, and in vivo derivedangiogenic factors may also be used. In some embodiments, thebiodegradable implant which contains said angiogenic factors contains acarrier. The carrier is preferably chosen so as to remain within theimplanted site for a prolonged period and slowly release the angiogenicfactors contained therein to the surrounding environment. This mode ofdelivery allows said angiogenic factors to remain in therapeuticallyeffective amounts within the site for a prolonged period. By providingsaid angiogenic factors within a carrier, the advantage of releasingsaid angiogenic factors directly into the target area is realized. Insome embodiments, the implant's carrier is provided in an injectableform. Injectability allows the carrier to be delivered in a minimallyinvasive and preferably percutaneous method. In some embodiments, theinjectable carrier is a gel. In others, the injectable carrier includeshyaluronic acid (HA).

In some embodiments, the carrier of the graft includes a porous matrixhaving an average pore size of at least approximately 25 micrometers.Preferably, the porous matrix has an average pore size of betweenapproximately 25 micrometers and approximately 110 micrometers. When theaverage pore size is in this range, it is believed that that porousmatrix will also act as a scaffold for in-migrating cells capable ofbecoming cells stimulatory of angiogenesis in the targeted area.Numerous examples of organic materials that can be used to form theporous matrix are known to one of skill in the art; these include, butare not limited to, collagen, polyamino acids, or gelatin.

The collagen source may be artificial (i.e., recombinant), orautologous, or allogenic, or xenogeneic relative to the mammal receivingthe implant. The collagen may also be in the form of an atelopeptide ortelopeptide collagen. Additionally, collagens from sources associatedwith high levels of angiogenesis, such as placentally derived collagen,may be used. Examples of synthetic polymers that can be used to form thematrix include, but are not limited to, polylactic acids, polyglycolicacids, or combinations of polylactic/polyglycolic acids. Resorbablepolymers, as well as non-resorbable polymers, may constitute the matrixmaterial. One of skill in the art will appreciate that the terms porousor semi-porous refer to the varying density of the pores in the matrix.

Scaffold structures may be used in some embodiments of the invention foranchoring or substantially causing adhesion between the implant andanatomical structures; the anatomical structures may be bone, cartilage,nerve, tendon, and ligament. In some embodiments, the method of adheringthe implant to the anatomical structures may be a gel. The gel, togetherwith the implant, can be injected to the graft site, in some embodimentsunder arthroscopic fluid conditions. The gel can be a biological orsynthetic gel formed from a bioresorbable or bioabsorbable material thathas the ability to resorb in a timely fashion in the body environment.

Suitable scaffold agents are also known to one of skill in the art, andmay include hyaluronic acid, collagen gel, alginate gel,gelatin-resorcin-formalin adhesive, mussel-based adhesive,dihydroxyphenylalanine-based adhesive, chitosan, transglutaminase,poly(amino acid)-based adhesive, cellulose-based adhesive,polysaccharide-based adhesive, synthetic acrylate-based adhesives,platelet rich plasma (PRP) gel, platelet poor plasma (PPP) gel, clot ofPRP, clot of PPP, Matrigel®, Monostearoyl Glycerol co-Succinate (MGSA),Monostearoyl Glycerol co-Succinate/polyethylene glycol (MGSA/PEG)copolymers, laminin, elastin, proteoglycans,poly(N-isopropylacrylamide), poly(oxyalkylene), a copolymer ofpoly(ethylene oxide)-poly(propylene oxide), polyvinyl alcohol andcombinations thereof.

In some embodiments, a pliable scaffold is preferred so as to allow thescaffold to adjust to the dimensions of the target site of implantation.For instance, the scaffold can include a gel-like material or anadhesive material, as well as a foam or mesh structure. Preferably, thescaffold can be a bioresorbable or bioabsorbable material. The scaffoldcan be formed from a polymeric foam component having pores with an opencell pore structure. The pore size can vary, but preferably, the poresare sized to allow tissue or angiogenic ingrowth. In some embodiments,the pore size is in the range of approximately 40 to approximately 900micrometers. The polymeric foam component can, optionally, include areinforcing component, such as, for example, woven, knitted, warpedknitted (i.e., lace-like), non-woven, and braided structures. In someembodiments where the polymeric foam component includes a reinforcingcomponent, the foam component can be integrated with the reinforcingcomponent such that the pores of the foam component penetrate the meshof the reinforcing component and interlock with the reinforcingcomponent. In some embodiments, the angiogenic growth factors arepredominantly released from a sustained delivery device by its diffusionthrough the sustained delivery device (preferably, through a polymer).In others, the angiogenic factors are predominantly released from thesustained delivery device by the biodegradation of the sustaineddelivery device (preferably, biodegradation of a polymer). In someembodiments, the implant includes a bioresorbable material whose gradualerosion causes the gradual release of the angiogenic factors. In someembodiments, the implant includes a bioresorbable polymer. Preferably,the bioresorbable polymer has a half-life of at least one month.Accordingly, in some embodiments, the implant includes the co-polymerpoly-DL-lactide-co-glycolide (PLG) admixed with the angiogenic factors.

In some embodiments, the implant essentially includes a hydrogel.Hydrogels can also be used to deliver said angiogenic factors in atime-release manner to the area of hypoperfusion. A “hydrogel,” asdefined herein, is a substance formed when an organic polymer (naturalor synthetic) is set or solidified to create a three-dimensionalopen-lattice structure that entraps molecules of water or other solutionto form a gel. The solidification can occur, e.g., by aggregation,coagulation, hydrophobic interactions, or cross-linking. The hydrogelsemployed in this invention rapidly solidify to keep said angiogenicfactors in proximity to either the blood vessel causative ofhypoperfusion, or the area associated with hypoperfusion. In someembodiments, said hydrogel is a fine, powdery synthetic hydrogel.Suitable hydrogels exhibit an optimal combination of such properties ascompatibility with the matrix polymer of choice, and biocompatibility.The hydrogel can include one or more of the following: polysaccharides,proteins, polyphosphazenes, poly(oxyethylene)-poly(oxypropylene) blockpolymers, poly(oxyethylene)-poly(oxypropylene) block polymers ofethylene diamine, poly(acrylic acids), poly(methacrylic acids),copolymers of acrylic acid and methacrylic acid, polyvinyl acetate, andsulfonated polymers.

In another embodiment of the invention, a direct injection of anangiogenic factor into the ischemic vertebral body can be performed toproduce angiogenesis within the vertebral body (and therefore thesubchondral capillary bed that supplies the disc with its nutrients).The vertebral pedicle, a route used in pedicle screw spinal implants aswell as vertebroplasty and kyphoplasty treatments of vertebralcompression fractures, can easily be entered with a direct catheter forinjection. The pedicle communicates with the vertebral body. Theinjection can be done percutaneously or with open surgery. Thisinjection can be short term (one injection) or be delivered within anindwelling catheter for longer administration. In addition, a devicecould be introduced through the pedicle that can be placed within thevertebral body for long term introduction of factor(s). In addition tothe vertebral pedicle, direct placement into the vertebral body throughthe vertebral body cortical wall could be a method of deliveringangiogenic factors to the vertebrae. This is performed at the time ofopen surgery or via the percutaneous route.

Embodiments of the invention are directed to detection ofischemia-associated osteoporosis and subsequent treatment throughangiogenic stimulation. While previous studies have demonstrated anassociation between atherosclerosis and osteoporosis, a causalrelationship was not identified. The invention discloses a noveldiagnostic algorithm that can be utilized in the diagnosis and selectionof patients for subsequent treatment utilizing pro-angiogenicapproaches. To date, a diagnostic imaging algorithm has not beendeveloped since no vascular basis for spinal disease has been acceptedin the field of spinal medicine and surgery. In one aspect of theinvention, magnetic resonance angiography (MRA), a special type of MRwhich creates three-dimensional reconstructions of vessels containingflowing blood, is utilized to identify vascular abnormalities. Byimaging the segmental arteries, a rating system is developed measuringthe amount of patency of the vessels. The following system is an exampleof such a system:

-   -   Arterial Occlusion (L1-L5): 2 vessels (left and right)    -   0=both vessels are patent    -   1=one vessel is stenotic    -   2=both vessels are stenotic    -   3=one vessel is occluded    -   4=one vessel is occluded and one stenotic    -   5=both vessels are occluded

In some embodiments of the invention, diffusion studies (DiffusionWeighted images or DWI) are performed for analyzing the diffusioncharacteristics of the disc and correlating it with disc degenerationand segmental artery stenosis.

In other embodiments, perfusion studies are performed using methods suchas Dynamic Contrast Enhanced MR Imaging for analysis of Perfusion of thevertebral bodies. Finally, combinations of imaging strategies are alsodisclosed in the current invention for generation of algorithms toinclude/exclude patients for the treatment of lower back pain, and/orvertebral osteoporosis.

Diffusion Studies

Diffusion Weighted Images (DWis) have mostly been used in the spine tohelp delineate benign and malignant vertebral collapse fractures(Griffith, J. F. et al. Radiology 236(3): 945-951, 2005). However, thetechnique appears to be well suited for research in analyzing thediffusion characteristics of the normal disc and correlating it withdisc degeneration (Rajasekaran, S. et al. Spine 29(23):2664-2667, 2004)and segmental artery stenosis and/or vertebral body perfusionabnormalities. Solute transfer into the central portion of the disc(nucleus) is dependent upon the concentration of the solute at thevertebral endplate (correlated with vascular perfusion) and thediffusion characteristics of the disc. Abnormalities in diffusioncontribute to disc degeneration. Analyzing diffusion properties amongvarious patient populations (as well as normal controls) may lead todata that can contribute to the ischemic disc disease diagnosis.

Perfusion Studies

Dynamic Contrast Enhanced MR Imaging for analysis of Perfusion of thevertebral bodies has been described using a 1.5 Tesla scanner in theevaluation of the possible ischemia-related osteoporosis (Laroche, M. etal. Clin Rheumatol 13:611-614, 1994; Chen, Wei-Tsung et al. Radiology220(1):213 2001; Shih, T. et al. Radiology 233(1):121-128, 2004).However, the correct imaging parameters on a 3 Tesla scanner have notbeen previously developed. The present inventors have determined imagingparameters for a 3 Tesla scanner. The images were acquired with aPhilips Achieva 3T system. For all imaging protocols, these were used:330 mm*300 mm FOV and a 6-element SENSE torso RF coil. The imagingsession started with the perfusion scan following the standardcalibration scans. A 3D FFE sequence with TRITE=3.5 ms/1.5 ms, SENSEfactor: 2.5(AP), 2(RL), flip angle=30.degree., with dynamic scan time of2.9 seconds was used and 7 slices in sagittal orientation with 6 mmthickness and 1.9 mm*1.9 mm pixel size were acquired. A total of 114volumes were collected, 2 of them before contrast injection. After thedynamic scans, T1 weighted anatomical images in sagittal plane werecollected using a TSE sequence with 0.5*0.5*3 mm.sup.3 voxel size. 14slices cover the same volume as dynamic scans. TR/TE=900 ms/10 ms, flipangle=90.degree. This was followed by a T2 weighted scan that hadidentical geometry to T1 scans and TR/TE=2940 ms/120 ms, flipangle=90.degree. Finally, contrast-enhanced angiography scans werecollected. Contrast bolus arrival was observed real-time using a single,50 mm thick coronal slice using FFE sequence in dynamic mode, collectingimages every 0.5 seconds. Once the contrast arrived in the descendingaorta, actual 3D angiography scan was started by the operatorimmediately. TR/TE=5.1 ms/1.78 ms, voxel size=0.8*0.8*1.5 mm.sup.3, withSENSE factor=4 was used to acquire 50 coronal slices. Segmental vesselson MRA were graded as occluded, stenotic or open. Discs were graded asper Pfirrmann (6). ROI-averaged time course (from whole vertebra and/orend-plate) was converted into a fractional enhancement time course andanalyzed using a compartmental model (Larsson, et al., MRM, 1996, 35:716-26; Workie, et al., MRI, 2004, 1201-10). The model fitting resultsin 6 parameters: Ktrans' (apparent volume transfer constant), kep (rateconstant), Vp′ (apparent fractional plasma volume), E (extractionfraction), tlag (arrival time of tracer in the ROI) and baseline.

Data were collected from control and experimental subjects to ascertainan “ischemic index” of the vertebral bodies. This is applied to futureresearch on ischemic/hypoxic disc disease. These data are correlatedwith the degree of disc degeneration and the degree of segmental arterystenosis to define a new clinical entity and the proper imaging toolsfor diagnosis of spinal, particularly lumbar, ischemia. Since perfusionmeasures the amount of blood supply coursing through the vertebralbodies and therefore the amount of nutrition available for the disc,this value may be important in developing treatment schemes based onimproving the blood supply to the vertebrae (and therefore, the disc).In addition, since the ROI (region of interest) can be placed anywhereon the scanned spine, the spinal cord can be investigated with thistechnique, providing another category of spinal disease (spinal cordinjury) with vascular anatomy imaging (MRA) and simultaneous DynamicPerfusion.

Spinal MR Spectroscopy

A loss of perfusion at the vertebral endplate results in less oxygenavailable for diffusion across into the nucleus of the disc. Sincesimple diffusion is the mechanism for solute transport within the discand not a pumping action (Soukane, M. D. et al. Journal of Biomechanics40:2645-2654, 2007), the oxygen concentration at the vertebral endplateis critical. Loss of oxygen (hypoxia) results in the chondrocytesshifting to anaerobic metabolism to produce energy. This inefficientprocess is associated with a shutdown in matrix production and resultingpoor matrix repair and maintenance (Homer, H et al. Spine 26:2543-2549,2001). High field strength spectroscopy (at least 3 Tesla) may beextremely important in the delineation of metabolic abnormalitiesassociated with ischemia within the intervertebral disc. It has beendemonstrated that lactate levels are elevated in discs dependent uponanaerobic metabolism. Therefore, lactate could be used as a biochemicalmarker signifying a disc that is “stressed” and at risk. In addition,low pH (associated with high lactate) has been demonstrated to be abiochemical mediator of discogenic pain. Other useful markers that maycorrelate with ischemia/hypoxia and the painful, degenerative discinclude, but are not limited to, determination of 31P levels as anindicator of energy level, water content as an indicator of proteoglycancontent and disc size. A larger disc indicates less efficientdistribution of oxygen and an increase in anaerobic metabolism.

Combination Imaging Strategies

Combining Imaging studies may provide important insight into thedescription of heretofore unknown vascular diseases of the spine. Inaddition, combining vertebral perfusion (of the vertebrae above andbelow the disc studied) with segmental artery stenosis and the degree ofdegenerative disc disease (and possibly diffusion and spectroscopy data)may describe a “new” etiology for subsets of patients with degenerativedisc disease and osteoporosis.

Either in combination or separate, growth factors, synthetic or treatedallograft or xenograft tissue for scaffold (or extra-cellular matrix)and stem cells may be utilized to “engineer” disc tissue with the goalof regenerating living tissue within the intervertebral disc. If thedegenerative disc to be treated requires that ischemia or hypoxiarelated causes need to be diagnosed and treated first or in combinationwith the tissue engineering techniques, then the diagnosis is ischemicdisc disease.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the invention and specificexamples provided herein without departing from the spirit or scope ofthe invention. Thus, it is intended that the invention covers themodifications and variations of this invention that come within thescope of any claims and their equivalents.

The following examples are for illustrative purposes only and are notintended, nor should they be interpreted to, limit the scope of theinvention.

EXAMPLES Example 1

Lumbar Segmental Artery Analysis Combined with Vertebral Body DynamicPerfusion

The vertebral arterial tree and vertebral body blood flow weresimultaneously evaluated for the purpose of vascular mapping of thelumbar spine. The goal of this study was (1) to develop a safe andreproducible technique of MRA and perfusion utilizing one injection ofcontrast, (2) to measure vertebral perfusion and compare intra-subjectand inter-subject results with the degree of segmental artery stenosisand degenerative disc disease, and (3) to begin evaluating normalcontrols.

Both the lumbar MRA and dynamic perfusion portions were contrastenhanced. Subjects were volunteers with degenerative disc disease (DDD)and low back pain and were referred from spine surgeon practices or byword of mouth. The images were acquired with a Philips Achieva 3Tsystem. For all imaging protocols, we used 330 mm*300 mm FOV and a6-element SENSE torso RF coil. The imaging session started with theperfusion scan following the standard calibration scans. A 3D FFEsequence with TR/TE=3.5 ms/1.5 ms, SENSE factor: 2.5(AP), 2(RL), flipangle=30.degree., with dynamic scan time of 2.9 seconds was used and 7slices in sagittal orientation with 6 mm thickness and 1.9 mm*1.9 mmpixel size were acquired. A total of 114 volumes were collected, 2 ofthem before contrast injection. After the dynamic scans, T1 weightedanatomical images in sagittal plane were collected using a TSE sequencewith 0.5*0.5*3 mm.sup.3 voxel size. Fourteen slices covered the samevolume as dynamic scans. TR/TE=900 ms/10 ms, flip angle=90.degree. Thiswas followed by a T2 weighted scan that had identical geometry to T1scans and TR/TE=2940 ms/120 ms, flip angle=90.degree. Finally, contrastenhanced angiography scans were collected. Contrast bolus arrival wasobserved real-time using a single, 50 mm thick coronal slice using FFEsequence in dynamic mode, collecting images every 0.5 seconds. Once thecontrast arrived in descending aorta, actual 3D angiography scan wasstarted by the operator immediately. TR/TE=5.1 ms/1.78 ms, voxelsize=0.8*0.8*1.5 mm.sup.3, with SENSE factor=4 was used to acquire 50coronal slices.

Segmental vessels on MRA were graded as occluded, stenotic or open.Discs were graded as per Pfirrmann (Pfirrmann, C. et al, Spine26:1873-1878, 2001). Region of interest (ROI)-averaged time course (fromwhole vertebra and/or end-plate) was converted into a fractionalenhancement time course and analyzed using a compartmental model(Larsson, et. al. MRM 35:716-726, 1996; Workie, et. al. MRI, 1201-1210,2004). The model fitting resulted in 6 parameters: K.sup.trans (apparentvolume transfer constant), k.sub.ep (rate constant), Vp′ (apparentfractional plasma volume), E (extraction fraction), tlag (arrival timeof tracer in the ROI) and baseline.

FIG. 1 shows Coronal MRA for a healthy control subject compared to 3subjects with symptoms of chronic lower back pain and DDD (Sub 1, Sub2,Sub3). The control was a 20-year old with normal discs and segmentalvessels from L1 to L5. The markers indicate areas of stenosis orocclusion. Sub 1 showed areas of stenosis indicated by the arrows. Sub2showed areas of occlusion indicated by the circle. Sub3 showed areas ofboth occlusion (left circle) and stenosis (right arrow).

Each of the three symptomatic subjects demonstrated at least onesegmental vessel that was either occluded or stenotic. Subjects 1 and 3demonstrated decreased K.sup.trans at the level of the vascularlesion(s) with subject 3 demonstrating an order of magnitude lower valueat all vertebral levels indicating a perfusion abnormality beyond theMRA identified lesions (FIG. 1). The other perfusion parameters(k.sub.ep, Vp and E) can be extracted from the acquired data and arehelpful in the interpretation. Pixel by pixel images can be generated ofany parameter (and through any slice) for visual comparison.

FIGS. 2A through 2D show the data for subject 3. Consistent with FIG. 1,the left arrow in Abnormal MRA Scan (FIG. 2A) shows a stenotic left L4segmental vessel. The right circle shows an occluded right L4 segmentalvessel. FIG. 2C depicts perfusion (K.sup.trans) showing that sub3 haslow blood flow through the vertebrae compared to the control subjectshown in FIGS. 3A through 3D where normal (higher) blood flow throughthe vertebrae is shown by the position of the side arrow in FIG. 3D.Scans such as those shown in FIGS. 2A through 3D can be color coded withthe color map demonstrating the disease visually and is more adaptablefor clinical use. Using this technique, data can be entered into apooled multicenter database. Subsets of patients that may have asignificant vascular and resultant ischemic/hypoxic component to theirdisease can then be identified and studied.

TABLE-US-00001 TABLE 1 K.sup.trans values of L3-51 vertebral bodies for3 subjects Vertebrae Control Sub 1 Sub 2 Sub 3 L3 0.215 0.146 0.1600.090 L4 0.220 0.148 0.156 0.084 L5 0.205 0.170 0.160 0.075 51 0.1490.160 0.146 0.053 K.sup.trans

Table 1 shows K.sup.trans values at sacral (51) and Lumbar (L3, L4, andL5) positions for the healthy control and three symptomatic subjects(sub1-3). Decreased K.sup.trans values are observed in all symptomaticsubject vertebral levels. K.sup.trans for sub3 is approximately 2-3 foldlower at each vertebra compared to sub1 and sub2.

FIG. 4A shows a Max Intensity projection (MIP) and FIG. 4B shows anAxial reconstruction for sub3. The circle in FIG. 4A and the right-sidecircle in FIG. 4B point to an occluded vessel. The left hand arrow inFIG. 4B points to a stenosis.

A method for studying the vascular anatomy and dynamics of the spine inone scanning session using a contrast agent is demonstrated. Spinalanatomy, vascular anatomy and sophisticated perfusion data can beobtained. K.sup.trans is the rate of transfer of contrast delivered tothe interstitial tissue, while the k.sub.ep is the rate the deliveredcontrast is cleared from the interstitial tissue, or “wash out”. Inaddition, E (the extraction fraction of contrast during its initialpassage within a given volume [ROI]) is another helpful parameter. Ifdecreased blood supply is an etiologic factor in a subset of patientswith DDD, this technique provides a mechanism by which investigators canstudy this disease in vivo.

Newer MR techniques such as MR Spectroscopy can be added to identifymetabolic abnormalities within the disc. For example, lactate, an endproduct of anaerobic metabolism, may be increased in the disc thatobtains its nutrients from vertebral bodies with poor perfusion. FIGS.5A and 5B demonstrate 2 discs that have undergone spectroscopy. FIG. 5Ashows the L4-5 disc (Disc A) and FIG. 5B shows the L5-1 disc (Disc B).When water is suppressed (FIG. 6B), Disc B has a higher lipid+lactatepeak [(Lip+Lac)/water=0.405] compared to Disc A [(Lip+Lac)/water=0.019],indicating a higher lactate concentration. This higher lactate may beassociated with anaerobic metabolism and discogenic pain.

Although the invention has been described and illustrated with a certaindegree of particularity, it is understood that the disclosure has beenmade only by way of example, and that numerous changes in the conditionsand order of steps can be resorted to by those skilled in the artwithout departing from the spirit and scope of the invention.

What is claimed is:
 1. A method of treating a patient having ischemictissue resulting from diminished microvascular blood flow comprising:identifying a patient with ischemic tissue resulting from diminishedmicrovascular blood flow; obtaining a composition comprising a carriersolution combined with an angiogenic growth factor, the carrier solutioncapable of adhering to an anatomical structure of the patient proximalto the ischemic tissue resulting from diminished microvascular bloodflow; and applying the composition to the anatomical structure of thepatient proximal to the ischemic tissue resulting from the diminishedmicrovascular blood flow, thereby inducing angiogenesis within at leasta portion of the ischemic tissue.
 2. The method of claim 1, wherein thecarrier solution comprises at least one of: a gel-like structure, acollagen, a member of the group consisting of a natural polymer, asynthetic polymer, a resorbable polymer, a non-resorbable polymer, abiodegradable polymer, a hydrogel, or a slow release polymer comprisingat least one angiogenic growth factor admixed therein.
 3. The method ofclaim 1, wherein the composition further comprises at least one memberof the group consisting of allograft tissue, synthetic allograft tissue,treated allograft tissue and xenograft tissue.
 4. The method of claim 1,wherein the angiogenic growth factor comprises at least one member ofthe group consisting of TPO, SCF, IL-1 to -11, flt-3L, G-CSF, GM-CSF,Epo, VEGF, FGF-1 to -23, EGF, IGF, NGF, LIF, PDGF, BMP, activin-A, PIGF,ephrins, endothelin-1, angiopoietin and HGF.
 5. The method of claim 1,wherein the carrier solution further comprises a chemoattractant capableof accelerating the process of angiogenesis and homing of at least oneof the members of the group consisting of endothelial cells andendothelial precursor cells.
 6. The method of claim 1, wherein thecomposition further comprises at least one member of the groupconsisting of embryonic stem cells, adult stem cells and progenitorcells.
 7. The method of claim 1, wherein the step of applying thecomposition to the anatomical structure of the patient proximal to theischemic tissue resulting from the diminished microvascular blood flowcomprises applying the composition to the anatomical structure of thepatient proximal to the ischemic tissue resulting from the diminishedmicrovascular blood flow a plurality of times over a time period of atleast a week.
 8. The method of claim 1, wherein the step of applying thecomposition to the anatomical structure of the patient proximal to, orinto, the ischemic tissue resulting from the diminished microvascularblood flow comprises injecting the composition into the anatomicalstructure of the patient proximal to the ischemic tissue resulting fromthe diminished microvascular blood flow.
 9. The method of claim 1,wherein the step of applying the composition to the anatomical structureof the patient proximal to, or into, the ischemic tissue resulting fromthe diminished microvascular blood flow comprises applying thecomposition to a surface of the anatomical structure of the patientproximal to the ischemic tissue resulting from the diminishedmicrovascular blood flow.
 10. The method of claim 1, wherein theanatomical structure of the patient comprises a blood vessel, a bonystructure, a cartilaginous structure, a nerve structure, a tendonstructure, a ligament structure, a soft tissue structure, or a discstructure of the patient.
 11. A method of treating a patient havingischemic tissue resulting from diminished microvascular blood flowcomprising: identifying a patient with ischemic tissue resulting fromdiminished microvascular blood flow; imaging a location in a patientwith the diminished microvascular blood flow; obtaining a compositioncomprising a carrier solution combined with a fibroblast growth factor-1(FGF-1), the carrier solution capable of adhering to an anatomicalstructure of the patient proximal to the ischemic tissue resulting fromdiminished microvascular blood flow; and applying the composition to theanatomical structure of the patient proximal to or in the ischemictissue resulting from the diminished microvascular blood flow, therebyinducing angiogenesis within at least a portion of the ischemic tissue.12. The method of claim 11, wherein the carrier solution comprises atleast one of: a gel-like structure, a collagen, a member of the groupconsisting of a natural polymer, a synthetic polymer, a resorbablepolymer, a non-resorbable polymer, a biodegradable polymer, a hydrogel,or a slow release polymer comprising at least one angiogenic growthfactor admixed therein.
 13. The method of claim 11, wherein thecomposition further comprises at least one member of the groupconsisting of allograft tissue, synthetic allograft tissue, treatedallograft tissue and xenograft tissue.
 14. The method of claim 11,wherein the composition further comprises at least one member of thegroup consisting of TPO, SCF, IL-1 to -11, flt-3L, G-CSF, GM-CSF, Epo,VEGF, FGF-2 to -23, EGF, IGF, NGF, LIF, PDGF, BMP, activin-A, PIGF,ephrins, endothelin-1, angiopoietin and HGF.
 15. The method of claim 11,wherein the carrier solution further comprises a chemoattractant capableof accelerating the process of angiogenesis and homing of at least oneof the members of the group consisting of endothelial cells andendothelial precursor cells.
 16. The method of claim 11, wherein thecomposition further comprises at least one member of the groupconsisting of embryonic stem cells, adult stem cells and progenitorcells.
 17. The method of claim 11, wherein the step of applying thecomposition to the anatomical structure of the patient proximal to theischemic tissue resulting from the diminished microvascular blood flowcomprises applying the composition to the anatomical structure of thepatient proximal to the ischemic tissue resulting from the diminishedmicrovascular blood flow a plurality of times over a time period of atleast a week.
 18. The method of claim 11, wherein the step of applyingthe composition to the anatomical structure of the patient proximal to,or into, the ischemic tissue resulting from the diminished microvascularblood flow comprises injecting the composition into the anatomicalstructure of the patient proximal to the ischemic tissue resulting fromthe diminished microvascular blood flow.
 19. The method of claim 11,wherein the step of applying the composition to the anatomical structureof the patient proximal to, or into, the ischemic tissue resulting fromthe diminished microvascular blood flow comprises applying thecomposition to a surface of the anatomical structure of the patientproximal to the ischemic tissue resulting from the diminishedmicrovascular blood flow.
 20. The method of claim 11, wherein theanatomical structure of the patient comprises a blood vessel, a bonystructure, a cartilaginous structure, a nerve structure, a tendonstructure, a ligament structure, a soft tissue structure, or a discstructure of the patient.